This application relates to the detection of cytotoxic substances and DNA damaging agents in whole effluent samples.
Detection of cytotoxic or DNA damaging agents in environmental samples (i.e., air, water, sewage or commercial effluent, or biological samples and the like) is an important aspect of pollution monitoring and control. In addition, the testing of control samples created in the laboratory is useful to assess the importance of any given material as a pollutant to be regulated or as a potential cytotoxic agent for pharmacological applications.
For many years, environmental testing and government regulation in the United States has focused on monitoring for discharge of individual chemical species. Recently, however, the US Environmental Protection Agency has recognized that this form of testing may be insufficient in some instances, and has approved so-called xe2x80x9cwhole effluent toxicityxe2x80x9d (WET) test procedures. Such tests are appropriate because effluent limitations on specific compounds do not necessarily provide adequate protection for aquatic life when the toxicity of effluent components is not known, effects of effluent components are additive, synergistic, or antagonistic, and/or when an effluent has not been chemically characterized.
The EPA manual (EPA/600/4-90/027F) describes tests for effluents and receiving waters, and includes guidelines for the following areas: Laboratory safety; quality assurance; facilities and equipment; effluent sampling and holding times; dilution water; test species selection, culturing, and handling; data collection, interpretation and utilization; report preparation; and dilutor and mobile toxicity test laboratory design. The acute toxicity tests in the manual generally involve exposure of any of 20 test organisms to each of five effluent concentrations and a control water. The test duration depends on the objectives of the test and the test species, and ranges from 24-96 hours. The manual includes a list of freshwater and marine test organisms, and specified test conditions for 10 commonly used freshwater and marine organismsxe2x80x94Cerodaphnia dubia, Daphnia magna, Daphnia pulex, fathead minnows (Pimephales promelas), rainbow trout (Oncorhynchus mykiss), brook trout (Saivelinus fontinalis), mysids (Mysidopsis bahia and Holmesimysis costata), Bannerfish shiners (Notropis leedsi), sheepshead minnows (Cyprinodon variegatus), and silversides (Menida menidia, M. beryllina, and M. peninsulae). The tests are used to determine the effluent concentration, expressed as a percent volume, that within the prescribed test period causes death in 50% the organisms (LC50), or whether survival in a given (single) concentration of effluent, or in receiving water, is significantly different than in controls. Where death is not easily detected, e.g., with some invertebrates like Ceriodaphnia and Daphnia (water fleas), immobilization is considered equivalent to death.
While the goal of evaluating whole effluent toxicity is a laudable one, the relatively advanced organisms used in the established testing procedures tend to make such procedures expensive to perform. Furthermore, because the testing is based upon a determination of the number of individuals that die, the statistics tend to be quantized (each organism is either dead or not dead) which means that very large numbers of organisms are required to get good statistical results. Furthermore, in the case of the simpler organisms, the methods require interpretation (is that Daphnia really not moving?) which may result in inconsistent results, and which requires a level of skill which increases the cost of implementing the test. Thus, it would be desirable to have a test methodology for whole effluent toxicity that makes use of an organism that is culturable, and that provides results which are unquantized, require less skill in interpretation and can be performed more frequently.
U.S. Pat. No. 5,387,508, which is incorporated herein by reference, describes a test for detection of cytotoxic substances in environmental samples using flagellate Tetramitus rostratus. These tests measure variations in cell division rate of the T. rostratus flagellates, as well as organism death and are applicable to solid, liquid or gaseous samples, including waste water samples. In each of the specific examples for analysis of a liquid sample (water or urine samples), the sample is first treated to concentrate organic compounds found in the sample by absorption on a resin, extraction and evaporation to dryness. Thus, there is no disclosure of a WET test in which all of the potentially toxic substances from the sample are evaluated in natural combination.
It has now been found that particle-feeding flagellate protozoa, including for example T. rostratus in the flagellate form, can in fact be used to determine whole effluent toxicity in liquid effluent samples, including water and sewage samples, without any requirement for concentration of the sample. Thus, in accordance with the present invention, there is provided a method for evaluating a sample for the presence of cytotoxic substances comprising the steps of:
(a) obtaining a sample for testing containing a plurality of potentially cytotoxic substances;
(b) combining the sample, either directly or after an optional filtration step, with a culture of particle-feeding flagellate; and
(c) monitoring the growth of the particle-feeding flagellate culture in the presence of the sample, wherein a decrease in growth of the culture in the presence of the sample is indicative of the presence of cytotoxic agents in the sample.